(1) An optimally flattened pH gradient was generated in electrofocusing through systematic variation of carrier constituent mixtures and selection of anolyte and catholyte. (2) Protein separation was demonstrated to be superior on flat pH gradients, compared to the separation on steep ones. (3) Biologically and immunologically active isohormones D and E of human growth were isolated in milligram amounts by electrofocusing. (4) Replacement of polyacrylamide in Steady-State Stacking by agarose as the anticonvectant was found to improve the purity of final fractionation products. (5) Isolation of biologically and immunologically active isohormones D and E of human growth hormone, in milligram amounts, was achieved by Isotachophoresis.